1. Field of the Invention
The present invention relates to an electrode plate for electrochemical measurements for detecting with high sensitivity and determining quantitatively a substance included in a living body in a slight amount.
2. Related Art
In recent years, electrode plates for electrochemical measurements for quantitatively determining the concentration of a saccharide such as sucrose, glucose or the like included in blood in a living body by a combination of a specific catalytic action of an enzyme and an electronic mediator having an electrode reaction activity.
According to such an electrode plate for electrochemical measurements, the reaction of a saccharide with an enzyme is utilized to quantitatively determine the concentration of the saccharide electrochemically. First, after a sample solution is prepared by mixing a blood sample with the enzyme and the electronic mediator, the enzymatic reaction is allowed between the saccharide and the enzyme. Thereafter, the electronic mediator coexisting therewith is electrochemically measured, whereby the saccharide included in the sample solution is quantitatively determined indirectly via the electronic mediator.
In this method, the enzymatic reaction is highly specific for the saccharide, accompanied by less influences from the temperature during operation, and the mechanism of the quantitative analysis unit is simple; therefore, ordinary persons can quantitatively determine the concentration of the saccharide in their own blood easily at home and the like by using this method.
The electrode plate for electrochemical measurements is suited for analyses of solution samples of a slight amount included in living bodies. Thus, applications of the electrode plate for electrochemical measurements have been attempted to sensors and the like through combining with a variety of organic materials or inorganic materials. The electrode response speed of the electrode plate for electrochemical measurements is accelerated as the area of a microelectrode carried by the electrode plate for electrochemical measurements is reduced. Therefore, various electrode shapes, and miniaturization of electrodes have been investigated.
However, as the area of the electrode is reduced, the resulting electric current value is lowered. For example, when the area of the electrode is miniaturized to approximately several hundred μm2, detectable electric current value may be lowered to several ten to several nA order. Thus, increase in noise response, and deterioration of the sensitivity may be caused in measurement. Accordingly, in order to avoid these defects, electrode plates for electrochemical measurements in which a plurality of microelectrodes are integrated were studied as in Japanese Patent No. 2556993 (column 6, FIG. 1, Patent Document 1), Japanese Patent No. 2564030 (column 7, FIG. 2, Patent Document 2), Japanese Unexamined Patent Publication No. 2006-78404 (column 25, FIG. 1, Patent Document 3) and Japanese Patent No. 3289059 (page 16, FIG. 5, Patent Document 4).
In Patent Documents 1 to 4, methods of producing a large quantity of microelectrodes on a substrate while keeping a constant distance between adjacent microelectrodes with favorable reproducibility are proposed.
FIG. 1A and FIG. 1B show a construction of a conventional electrode plate for electrochemical measurements disclosed in Patent Document 1.
This electrode plate for electrochemical measurements 10 is constructed by laminating insulative substrate 1/bottom electrode 2 that functions as an oxidation electrode/insulating layer 3/surface electrode 4 that functions as a reduction electrode. A large number of cylindrical micropores 5 are formed on the surface of the surface electrode 4, and the surface of the bottom electrode 2 is exposed to the micropore 5.
The insulative substrate 1 is constituted with, for example, a silicon substrate with an oxide film, generally referred to, in which oxide film 1b is adhered on the main surface of silicon substrate 1a. The bottom electrode 2 is an oxidation electrode formed with a metal, a semimetal, a carbonic material, or a semiconductor on the surface of the oxide film 1b of the substrate 1 (i.e., insulator surface). The surface electrode 4 is a reduction electrode formed with a metal, a semimetal, or a semiconductor on the insulating layer 3, similarly to the bottom electrode 2. A working electrode pair is formed with the bottom electrode 2 and the surface electrode 4. In other words, both the bottom electrode 2 and the surface electrode 4 function as working electrodes, and more specifically, the bottom electrode 2 functions as an oxidation electrode, while the surface electrode 4 functions as a reduction electrode, as described above. In FIG. 1A and FIG. 1B, the reference numeric character 7 represents an opening for drawing the electrode, opened so as to connect an outer lead to one end of the bottom electrode 2. Herein, the micropore represents a hole that completely penetrates through the insulating layer 3 and the surface electrode 4, and then reaches to the surface of the bottom electrode 2.
In an apparatus for electrochemical measurements in which the electrode plate for electrochemical measurements as described above is used, a potential is applied between the bottom electrode 2 and the surface electrode 4 for achieving an electric current response. When the apparatus for electrochemical measurements is constructed with three electrodes, i.e., bottom electrode 2, surface electrode 4, and a counter electrode (not shown in the Figure), a potential is applied between the bottom electrode 2 and the counter electrode, and between the surface electrode 4 and the counter electrode, provided that the potential shown by the counter electrode in the sample solution is zero. In addition, when the apparatus for electrochemical measurements is constructed with four electrodes, i.e., bottom electrode 2, surface electrode 4, a reference electrode (not shown in the Figure), and an auxiliary electrode (not shown in the Figure), a potential is applied between the bottom electrode 2 and the reference electrode, and between the surface electrode 4 and the reference electrode, provided that the potential shown by the reference electrode in the sample solution is zero.
In Patent Document 4 and J. Electrochem. Soc., Vol. 138, No. 12, page 3551 (1991)(Nonpatent Document 1), an electrode plate for electrochemical measurements is proposed in which cylindrical micropores 5 are provided such that the intervals among them becomes greater than their diameter, and the results of electrochemical measurements using the same are reported. In these Documents, the surface electrode 4 that is a macroelectrode has an area greater than the bottom electrode 2 that is an assembly of microelectrodes. Upon measurement, potentials are applied, respectively, which can cause an oxidative reaction on the bottom electrode 2, and a reductive reaction on the surface electrode 4. It is reported that self-induced redox cycle is thus generated between the bottom electrode 2 and the surface electrode 4, whereby apparently high electric current response can be achieved.
In this manner, a target substance such as a saccharide is quantitatively determined via an electronic mediator that is present in a sample solution.
Alternatively, even though a potential that causes a reductive reaction is applied on the bottom electrode 2, while a potential that causes an oxidative reaction is applied on the top electrode 4, similar self-induced redox cycle is generated.
Hereinbelow, the self-induced redox cycle described in Patent Document 4, and Nonpatent Document 1 and Koichi Aoki et al., “Electrochemical Measurement Method Using Microelectrode” edited by The Institute of Electronics, Information and Communication Engineers, published on Feb. 10, 1998 pages 48-49 and 70-71 (Nonpatent Document 2) are explained with reference to FIG. 2.
The self-induced redox cycle in FIG. 2 proceeds on two working electrodes, i.e., microelectrode 21 and macroelectrode 22.
An oxidative reaction of reductant 23 is caused to produce oxidant 24 on the surface of the microelectrode 21, whereby an oxidation current flows to the microelectrode 21.
On the surface of a part 22a, which is close to the microelectrode 21, of the macroelectrode 22, the oxidant 24 is reduced to be converted into reductant 25, whereby a reductive electric current flows to the macroelectrode 22.
Furthermore, the reductant 25 is diffused to reach to the surface of the microelectrode 21, whereby an oxidative reaction is caused again from the reductant 23 to the oxidant 24, leading to an oxidation current to flow toward the microelectrode 21. As a consequence, the reductant 23 can be fed to the surface of the microelectrode 21 by reducing the oxidant 24 generated from the microelectrode 21 to give the reductant 25 on the surface of the macroelectrode 22a. 
Accordingly, as a result of occurrence a so-called redox cycle reaction in which an oxidative reaction and a reductive reaction recur between the microelectrode 21 and the macroelectrode 22a, an electric current constantly flows to the microelectrode 21, and thus the target substance included in a sample solution in a slight amount can be detected and quantitatively determined.
Moreover, in order to improve the efficacy of the measurement with high sensitivity, electrode pairs consisting of an oxidation electrode and a reduction electrode by which a redox cycle proceeds are formed as many as possible through forming a larger number of the microelectrodes 21 on the substrate.